Interaction of rifalazil with oxidant-generating systems of human polymorphonuclear neutrophils.

It is well acknowledged that ansamycins display immunosuppressive and anti-inflammatory properties in vitro and in vivo. Rifalazil, a new ansamycin derivative, has not been studied in the context of inflammation. In particular, there are no data on the possible interference of rifalazil with oxidant production by phagocytes. We have compared the antioxidant properties of rifalazil to those of rifampin, a drug well known in this context, by using cellular and acellular oxidant-generating systems. Oxidant production by polymorphonuclear neutrophils was measured in terms of cytochrome c reduction, lucigenin-amplified chemiluminescence (Lu-ACL), and the 2',7'-dichlorofluorescin diacetate H2 (DCFDA-H2) technique (intracellular oxidant production). Rifalazil impaired O2- production in a concentration-dependent manner, with 50% inhibitory concentrations (IC50) (concentrations which inhibit 50% of the response) of 5.4 (30 and 60 min of incubation) and 6.4 (30 min) mg/liter, respectively, for phorbol myristate acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. In agreement with the published fMLP-like activity of rifampin, the inhibitory effect of rifampin was significantly greater for fMLP (IC50 of 5.6 mg/liter) than for PMA (IC50 of 58 mg/liter) stimulation. Alteration of intracellular oxidant production was also observed with IC50 values similar to those obtained by the cytochrome assay. In addition, rifalazil and rifampin (> or = 25 mg/liter) scavenged O2-, as demonstrated by the acellular (hypoxanthine-xanthine oxidase) system. Interference with light detection systems was evidenced for both drugs by Lu-ACL. The clinical relevance of the antioxidant effect of rifalazil demonstrated in vitro, in particular its potential anti-inflammatory activity, requires further investigation.

Accumulation of azithromycin and roxithromycin in tracheal epithelial fetal cell lines expressing wild type or mutated cystic fibrosis transmembrane conductance regulator protein (CFTR).

Macrolides are accumulated in phagocytes, partially via an active transport system; the membrane carrier is not identified but many data indicate a link with the P-glycoprotein family which includes the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. We have used two epithelial cell lines which express either wild-type (N cells) or mutated (homozygous deltaF508) (F cells) CFTR to study the cellular accumulation of two macrolides (azithromycin and roxithromycin). Adherent cells were incubated with the radiolabeled drugs before extensive washings and counting. Azithromycin was better (about 2-fold) accumulated in F cells up to 60 min but then plateaued, whereas accumulation continued without saturation over 3 hours in N cells. Roxithromycin was also better (1.5-fold) accumulated in F cells at 15 and 30 min, but there were no differences at further incubation times. Macrolide efflux from loaded N and F cells, and the susceptibilities of the carrier systems (entry and efflux) to various pharmacologic agents were similar to those previously observed with phagocytes. These data suggest that the macrolide carriers (for entry and efflux) are not strictly specific for phagocytes and that the CFTR protein plays a role in macrolide uptake.

Interaction of macrolides and ketolides with the phagocytic cell line PLB-985.

Interactions between antibacterial agents and polymorphonuclear neutrophils (PMNs) are a major focus of investigation. Owing to the variable drug susceptibility of PMNs from different individuals, in vitro studies require samples from large panels of healthy volunteers to reach statistical significance. Here, we used a phagocytic cell line, PLB-985, which can differentiate into mature PMNs in vitro, for the study of cellular interactions (drug uptake and antioxidant effects) of two macrolides (azithromycin and roxithromycin) and four ketolides [HMR 3004, HMR 3647 (telithromycin), HMR 3562 and HMR 3787]. The oxidative burst of differentiated (D) cells was inhibited by macrolides and ketolides. IC50% values (concentrations impairing the oxidative burst by 50%), determined after 30 min of incubation, were as follows for azithromycin, roxithromycin, HMR 3004, telithromycin, HMR 3562 and HMR 3787, respectively: 40, 39, 15, 23, 26, and 33 mg/l (fMLP stimulation) and 37, 86, 39, 43, 14, and 31 mg/l (PMA stimulation). These values were similar to those obtained with PMNs. Uptake of the two macrolides was significantly lower in non-differentiated (ND) cells than in D cells and PMNs. The cellular/extracellular (C/E) concentration ratios at 60 min for PMNs, D and ND PLB were respectively 67, 25 and 11 (roxithromycin) and 159, 137 and 48 (azithromycin). Ketolide uptake by ND-PLB was also significantly lower than that obtained with PMNs (C/E ratios at 60 min were about 75 versus 265 (HMR 3004), 36 vs 230 (telithromycin), 75 vs 235 (HMR 3562) and 20 vs 130 (HMR 3787). Although the active carrier system seemed to be present in ND cells, its activation pathway was not functional. Thus, the PLB-985 cell line is a good in vitro model for studying drug-PMN interactions. The use of ND and D cells may shed light on the nature and activation pathways of macrolide transport systems present on the PMN membrane.

Structure-activity relationships among 9-N-alkyl derivatives of erythromycylamine and their effect on the oxidative burst of human neutrophils in vitro.

Macrolide antibiotics have recently triggered much interest owing to the immunomodulatory potential of some derivatives, particularly in the field of inflammatory diseases. Among the possible mechanisms underlying these anti-inflammatory effects, macrolide-induced inhibition of oxidant production by phagocytes has attracted much attention. We and others have previously reported that erythromycin A-derived macrolides impair the phagocyte oxidative burst, a property linked to the presence of L-cladinose. However, we have also demonstrated that other substituents can be involved in the modulation of phagocyte function. Here we have extended the analysis of structure-activity relationships by studying the effects of five 9-N-alkyl derivatives of erythromycylamine on oxidant production by human neutrophils in vitro. LY211397 (2-methoxyethyl derivative) neither altered cell viability nor superoxide anion production. LY281389 (n-propyl derivative) did not alter cell viability and was slightly more inhibitory than erythromycylamine for the production of superoxide anion; its IC50 (concentration that inhibits 50% of the neutrophil response) was about 18 and 24 microM (versus 72 and 74 pM for erythromycylamine) after 60 min of incubation following fMLP and PMA stimulation, respectively. LY80576 (N-phenyl-3-indolylmethyl derivative), LY281981 (3-phenyl-n-propyl derivative) and LY57843 (benzyl derivative) all displayed cellular toxicity at high pharmacological concentrations after 30 to 60 min of incubation. Interestingly, these latter three drugs exhibited a rapid (5 min incubation) and strong inhibitory effect on the neutrophil oxidative burst from either stimulus, with IC50 values of 3 to 10 pM. Further in-vitro and in-vivo investigations are required to analyze the anti-inflammatory potential of these three derivatives.

Effect of telithromycin (HMR 3647) on polymorphonuclear neutrophil killing of Staphylococcus aureus in comparison with roxithromycin.

HMR 3647 (telithromycin), a new ketolide, is active on intracellular pathogens. It was previously demonstrated that it inhibits superoxide anion production in a time- and concentration-dependent manner, at concentrations which inhibit 50% of the control response of about 55 microg/ml (5 min) to 30 microg/ml (30 min); these values are similar to those obtained with roxithromycin, a classical erythromycin A derivative. Here we investigated whether these drugs modified the bactericidal activity of human polymorphonuclear neutrophils (PMN) on four strains of Staphylococcus aureus with different profiles of susceptibility to macrolides and ketolides. We found that the main factor involved in killing was the antibacterial potency of the drugs, although combinations of antibiotics with PMN were slightly more active than each component used alone against two of the four strains. In addition, high concentrations of the drugs, which impaired the PMN oxidative burst, did not impair PMN bactericidal activity. Likewise, some cytokines which enhance PMN oxidative metabolism did not modify PMN bactericidal activity in the presence or absence of macrolides or ketolides. These data suggest that oxygen-independent mechanisms contribute to the bactericidal activity of PMN on these strains of S. aureus. Both live and/or heat-killed bacteria impaired the uptake of telithromycin and roxithromycin (but not that of levofloxacin, a quinolone) in a concentration-dependent manner, owing to a modulation of PMN transductional systems involved in the activation of the macrolide carrier.

Cellular uptake of two fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils in vitro.

We analyzed the cellular accumulation of two new fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils (PMN) in vitro. Both compounds were rapidly taken up by PMN, with a cellular-to-extracellular concentration ratio (C/E) of about 141 (HMR 3562) and 117 (HMR 3787) at 5 min, and this was followed by a plateau at 60 to 180 min, with a C/E of >300 at 180 min. Both ketolides were mainly located in PMN granules (about 75%) and egressed slowly from loaded cells (about 40% at 60 min), owing to avid reuptake. Uptake was moderately sensitive to external pH, and activation energy was also moderate (about 70 kJ/mol). As with other macrolides and ketolides, the existence of an active transport system was suggested by (i) the strong interindividual variability in uptake kinetics, suggesting variability in the number or activity of a transport protein; (ii) the saturation kinetics characteristic of a carrier-mediated transport system (V(max), about 2,300 ng/2.5 x 10(6) PMN/5 min; K(m), about 50 microg/ml); (iii) the inhibitory effects of Ni(2+) (a blocker of the Na+-Ca(2+) exchanger), phorbol myristate acetate (a protein kinase C activator), and H89 (a protein kinase A inhibitor). Although these two ketolides are more related to HMR 3647 (telithromycin), it is interesting that the presence of a fluoride gave these molecules a cellular pharmacokinetics more like those of HMR 3004 than those of HMR 3647. The macrolide transport system has not been yet elucidated, but our data confirm that, despite variations in chemical structure, all erythromycin A derivatives share a transmembrane transport system.

Immunomodulation by macrolide antibiotics.

Macrolide antibiotics are strongly concentrated within host cells, a property that sustains their activity against intracellular pathogens and is likely responsible for the modulation of cell metabolism and function. There is extensive literature on the subject of macrolide-induced modulation of immune responses. Erythromycin A derivatives seem to display anti-inflammatory activity in vitro, in some animal models and in various clinical settings such as diffuse panbronchiolitis (DPB). The underlying mechanisms are not yet fully understood: inflammatory cytokine and oxidant production by phagocytes is down-regulated by these drugs, but other possible targets include bacterial virulence factors, bronchial and epithelial cells, etc. Also, a link has been suggested between the macrolide transmembrane carrier system and the P-glycoprotein family, which comprises MDR (multiple drug resistance) and CFTR (cystic fibrosis transmembrane conductance regulator), which are respectively involved in the chemotherapeutic resistance of cancer cells and in the genesis of cystic fibrosis.

Interference of antibacterial agents with phagocyte functions: immunomodulation or "immuno-fairy tales"?

Professional phagocytes (polymorphonuclear neutrophils and monocytes/macrophages) are a main component of the immune system. These cells are involved in both host defenses and various pathological settings characterized by excessive inflammation. Accordingly, they are key targets for immunomodulatory drugs, among which antibacterial agents are promising candidates. The basic and historical concepts of immunomodulation will first be briefly reviewed. Phagocyte complexity will then be unravelled (at least in terms of what we know about the origin, subsets, ambivalent roles, functional capacities, and transductional pathways of this cell and how to explore them). The core subject of this review will be the many possible interactions between antibacterial agents and phagocytes, classified according to demonstrated or potential clinical relevance (e.g., neutropenia, intracellular accumulation, and modulation of bacterial virulence). A detailed review of direct in vitro effects will be provided for the various antibacterial drug families, followed by a discussion of the clinical relevance of these effects in two particular settings: immune deficiency and inflammatory diseases. The prophylactic and therapeutic use of immunomodulatory antibiotics will be considered before conclusions are drawn about the emerging (optimistic) vision of future therapeutic prospects to deal with largely unknown new diseases and new pathogens by using new agents, new techniques, and a better understanding of the phagocyte in particular and the immune system in general.

Effect of proinflammatory cytokines on the interplay between roxithromycin, HMR 3647, or HMR 3004 and human polymorphonuclear neutrophils.

Cytokines, the hallmarks of infectious and inflammatory diseases, modify phagocyte activities and thus may interfere with the immunomodulating properties of antibacterial agents. We have investigated whether various proinflammatory cytokines (interleukin 1 [IL-1], IL-6, IL-8, gamma interferon, tumor necrosis factor alpha [TNF-alpha], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) modify two macrolide properties, i.e., inhibition of oxidant production by polymorphonuclear neutrophils (PMN) and cellular uptake. Roxithromycin and two ketolides, HMR 3647 and HMR 3004, were chosen as the test agents. TNF-alpha and GM-CSF (but not the other cytokines) decreased the inhibitory effect of HMR 3647 only on oxidant production by PMN. Fifty percent inhibitory concentrations were, however, in the same range in control and cytokine-treated cells (about 60 to 70 microgram/ml), suggesting that HMR 3647 acts downstream of the priming effect of cytokines. In contrast, the impairment of oxidant production by roxithromycin and HMR 3004 was unchanged (or increased) in cytokine-treated cells. This result suggests that HMR 3004 (the strongest inhibitory drug, likely owing to its quinoline side chain) and roxithromycin act on a cellular target upstream of cytokine action. In addition, TNF-alpha and GM-CSF significantly (albeit moderately) impaired (by about 20%) the uptake of the three molecules by PMN. The inhibitory effect of these two cytokines seems to be related to activation of the p38 mitogen-activated protein kinase. Our data also illuminate the mechanism underlying macrolide uptake: protein kinase A- and tyrosine kinase-dependent phosphorylation seems to be necessary for optimal uptake, while protein kinase C activation impairs it. The relevance of our data to the clinical setting requires further investigations, owing to the complexity of the cytokine cascade during infection and inflammation.

Mechanism underlying levofloxacin uptake by human polymorphonuclear neutrophils.

The mechanism of radiolabeled levofloxacin ([3H]levofloxacin) uptake by human polymorphonuclear neutrophils (PMNs) was investigated by a classical velocity centrifugation technique. PMNs were incubated with levofloxacin for 5 to 180 min under various conditions before centrifugation through an oil cushion. Radioactivity was measured in the cell pellet to determine the amount of cell-associated drug. The uptake of levofloxacin was moderate with a cellular concentration/extracellular concentration ratio of about 4 to 6. Levofloxacin accumulated in PMNs parallel to the extracellular concentration, without saturation, over the range of 2.5 to 200 mg/liter (linear regression analysis: r = 0.92; P < 0.001). The activation energy was low (36 +/- 7.2 kJ/mol). Levofloxacin uptake was increased in Ca(2+)-depleted, EGTA-containing medium by approximately 33% (P = 0.022), while Ni2+, a Ca2+ channel inhibitor, inhibited it in a concentration-dependent manner, with the concentration that inhibited 50% of control uptake being approximately 2.65 mM. Verapamil (an L-type Ca2+ channel inhibitor) and other pharmacologic agents which modify Ca2+ homeostasis did not modify levofloxacin uptake. Interestingly, Ca2+ and Mg2+ inhibited levofloxacin uptake in a concentration-dependent manner. EGTA, Ni2+, and verapamil did not modify levofloxacin efflux; thapsigargin, a Ca2+ pool-releasing agent, modestly increased the intracellular retention of levofloxacin. In addition, contrary to other fluoroquinolones, probenecid at 1 to 10 mM did not modify either levofloxacin uptake or efflux. These data are consistent with a mechanism of passive accumulation of levofloxacin in PMNs. Extracellular Ca2+ and Mg2+ may influence the structural conformation of levofloxacin or the lipophilicity of PMN membranes, thus explaining their effect on levofloxacin uptake.

Interactions between HMR 3647, a new ketolide, and human polymorphonuclear neutrophils.

HMR 3647, a new ketolide, is active upon intracellular pathogens. We previously demonstrated that HMR 3004 (RU 64004), another ketolide, is highly concentrated by human polymorphonuclear neutrophils (PMNs). This prompted us to evaluate whether the presence of a 3-keto group instead of an L-cladinose, a neutral sugar characteristic of erythromycin A derivatives, confers peculiar pharmacokinetic properties with regard to cellular accumulation and efflux. After incubation with the radiolabelled drug, HMR 3647 uptake was determined by a velocity gradient centrifugation technique. HMR 3647 was avidly concentrated by PMNs, without saturation, over a 3-h incubation period, with cellular-to-extracellular concentration ratios of 31 +/- 4.2 at 5 min and up to 348 +/- 27.1 at 180 min. About 60% of HMR 3647 was located in the granular compartment; less than 6% was associated with the membranes. HMR 3647 gradually egressed from loaded cells placed in drug-free medium. Uptake was dependent on environmental temperature (activation energy, 128 +/- 9. 4 kJ/mol) but not on extracellular pH. HMR 3647 displayed Michaelis-Menten saturation kinetics with a mean Vmax of 2315 ng/2.5 x 10(6) PMNs/5 min and a mean Km of 117 mg/liter (144 microM). As already observed with erythromycin A-derived macrolides, extracellular Ca2+ was necessary for optimal uptake of HMR 3647. Interestingly, verapamil increased the uptake of HMR 3647 at 5 min, but this was followed by gradual inhibition at later incubation times, a phenomenon probably related to stimulation of drug efflux. The impact of intracellular accumulation of HMR 3647 on PMN functions was also investigated. In contrast to other erythromycin A derivatives, HMR 3647 only weakly triggered granule exocytosis, but it inhibited superoxide anion production in a time- and concentration-dependent manner, with concentrations which inhibited 50% of control response of 55 (67 microM) (5 min) and 30 (36 microM) (30 min) mg/liter for formyl-methionyl-leucyl-phenylalanine stimulation and 117 (143 microM) (5 min) and 44 (54 microM) (30 min) mg/liter for phorbol myristate acetate stimulation.

Antibacterial agents--phagocytes: new concepts for old in immunomodulation.

The possibility that antibacterial agents, primarily directed against microorganisms, also modify host functions is widely recognized. While a knowledge of these non-antimicrobial effects of antibiotics, sometimes considered as 'side-effects', is necessary to prevent antibiotic-associated toxicity, the development of drugs derived from antibacterial agents for use in non-infectious diseases (e.g. motilins and antidiabetic drugs) is a new field of therapeutic research. Interactions between antibacterial drugs and the immune system may contribute to therapeutic efficacy in infectious diseases [1,2]. The immune system itself is a complex pyramid of redundant cellular factors/humoral effectors/mediators, whose fine regulation is just beginning to be unraveled. Phagocytes, ubiquitous and multifaceted cells are key components of cellular immunity, being involved both in immediate defences against non-self targets (pathogens, tumour cells, exogenous molecules, etc.) and in the regulation and triggering of specific immune responses. They are thus, prime targets of immune response modifiers. This review reconsiders the widely explored problem of interactions between antibacterial agents and phagocytes, focusing on future prospects in both infectious and non-infectious diseases.

Anti-inflammatory activity of macrolides: a new therapeutic potential?

The important role played by macrolides in the chemotherapy of infectious diseases is well established, but there is still much speculation about their anti-inflammatory potential. A review of in-vitro and ex-vivo studies reported in the literature shows that macrolides have potentially relevant immunomodulatory effects. In-vitro data suggest that erythromycin A derivatives have a direct effect on neutrophil function and the production of cytokines involved in the inflammation cascade. The ex-vivo results indicate that short-term administration of macrolides may enhance the immune response while long-term administration results in immunosuppression. Further research is required to improve our understanding of the therapeutic activity of macrolides.

Immunological effects of macrolides.

Various reviews have highlighted the potential immuno-modulating properties of macrolides. Recent data in this field raise the possibility of new therapeutic prospects in cancer and inflammatory diseases (cystic fibrosis, asthma, atherosclerosis, etc.). Advances have also been made in our understanding of the interactions between macrolides and host immune effectors, particularly phagocytes. The third millennium should see exciting new uses of macrolides.

Erythromycin A-derived macrolides modify the functional activities of human neutrophils by altering the phospholipase D-phosphatidate phosphohydrolase transduction pathway: L-cladinose is involved both in alterations of neutrophil functions and modulation of this transductional pathway.

All erythromycin A derivatives, irrespective of the size of the lactone ring and the nature of the substituent, inhibit oxidant production by neutrophils and promote their degranulation. We demonstrate in this study that the L-cladinose at position 3 of the lactone ring is a key structure in the modulation of these two neutrophil functions, suggesting that this sugar (alone or combined with a lactone structure) interferes with cell target(s) involved in both oxidant production and exocytosis. Taking roxithromycin as an example of erythromycin A derivatives, we also found that these molecules interfered with the phospholipase D (PLD)-phosphatidate phosphohydrolase pathway in two ways. In nonstimulated neutrophils, roxithromycin and all L-cladinose-bearing molecules activated PLD, as reflected by 1-O-[3H]alkyl-2-acyl-phosphatidyl-ethanol production. In addition, these drugs induced an accumulation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA), but not 1-O-[3H]alkyl-2-acylglycerol. PA accumulation seems to be involved in the induction of exocytosis by macrolides, as the roxithromycin-induced release of granular enzymes was impaired strongly in the presence of ethanol. By contrast, in stimulated neutrophils, roxithromycin inhibited PLD activity and totally impaired 1-O-[3H]alkyl-2-acylglycerol production. The inhibition of diglyceride production by roxithromycin (not its descladinosyl derivative) could explain its inhibitory effect on oxidant production. The relevance of our data to the clinical situation, particularly the anti-inflammatory activity of these drugs, requires further investigation.

Cellular accumulation of the new ketolide RU 64004 by human neutrophils: comparison with that of azithromycin and roxithromycin.

We analyzed the uptake of RU 64004 by human neutrophils (polymorphonuclear leukocytes [PMNs]) relative to those of azithromycin and roxithromycin. RU 64004 was strongly and rapidly accumulated by PMNs, with a cellular concentration/extracellular concentration ratio (C/E) of greater than 200 in the first 5 min, and this was followed by a plateau at 120 to 180 min, with a C/E of 461 +/- 14.8 (10 experiments) at 180 min. RU 64004 uptake was moderately sensitive to external pH, and activation energy was also moderate (63 +/- 3.8 kJ/mol). RU 64004 was mainly located in PMN granules (about 70%) and egressed slowly from loaded cells, owing to avid reuptake. The possibility that PMN uptake of RU 64004 and other macrolides occurs through a carrier-mediated system was suggested by three key results. First, there existed a strong interindividual variability in uptake kinetics, suggesting variability in the numbers or activity of a transport protein. Second, macrolide uptake displayed saturation kinetics characteristic of that of a carrier-mediated transport system: RU 64004 had the highest Vmax value (3,846 ng/2.5 x 10(6) PMNs/5 min) and the lowest Km value (about 28 microM), indicating a high affinity for the transporter. Third, as observed previously with other erythromycin A derivatives, Ni2+ (a blocker of the Na+/Ca2+ exchanger which mediates Ca2+ influx in resting neutrophils) impaired RU 64004 uptake by PMNs, with a 50% inhibitory concentration of about 3.5 mM. In addition, we found that an active process is also involved in macrolide efflux, because verapamil significantly potentiated the release of all three macrolides tested. This effect of verapamil does not seem to be related to an inhibition of Ca2+ influx, because neither EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N',N'-tetraacetic acid] nor Ni2+ modified macrolide efflux. The nature and characteristics of the entry- and efflux-mediating carrier systems are under investigation.

The prohost effect of antimicrobial agents as a predictor of clinical outcome.

Apart from their direct antimicrobial activity, some antimicrobial agents may interfere in the complex host-microorganism interplay by modulating the natural response to invading pathogens. The three main aspects of this non-antibiotic effect (alteration of bacterial virulence, synergism with/impairment of the natural host response, impact on effector cell progenitors) will be briefly reviewed here, along with their potential incidence in the clinical outcome of infectious diseases.

[Intracellular penetration of macrolides]. / Pénétration intracellulaire des macrolides.

CELL PENETRATION: One of the main features of macrolides is their capacity to penetrate host cells. This property is the basis of their action against intercellular microorganisms and can explain characteristic pharmacokinetics as well as interference with cell metabolism. INTRACELLULAR CONCENTRATION: Erythromycin A derivatives with a single base group (roxithromycin) accumulate rapidly to a saturation point both the cytoplasm and in granulations. Intracellular concentrations of derivatives with two base groups do not reach saturation due to slow exflux. THERAPEUTIC ACTION: Because of their intracellular concentration, macrolides are indicated for the treatment of cell-associated pathogens. Exflux allows possible selective transfer into infected tissues. Macrolides also have an effect on functional activity of the host cells.

Comparison of various macrolides on stimulation of human neutrophil degranulation in vitro.

Macrolide antibiotics are taken up and concentrated by host cells, particularly phagocytes, and are likely candidates to modify cell functions. In this study, we extended our previous work concerning the effect of three 14-membered-ring macrolides (dirithromycin, erythromycin and erythromycylamine) on human neutrophil exocytosis, and found that three other erythromycin A derivatives (roxithromycin, clarithromycin and the azalide, azithromycin) also triggered neutrophil degranulation in a time- and concentration-dependent manner. After 30 min of incubation, the correlation coefficients for concentration-dependence for roxithromycin were 0.885, 0.739 and 0.750 (P < 0.005) and for clarithromycin were 0.795, 0.599, 0.733 (P < 0.02), respectively, for lysozyme, beta-glucuronidase and lactoferrin release. Although the underlying mechanism was not elucidated, these and previous data suggest that intracellular accumulation is a prerequisite. Furthermore, comparison of the characteristics of macrolide-induced exocytosis with those of exocytosis triggered by the synthetic chemotactic stimulus FMLP suggested that different mechanisms are involved. In keeping with this possibility, we showed that combined treatment (macrolides plus FMLP) resulted in totally additive exocytosis of azurophilic but not specific granules. The clinical relevance of our data remains to be ascertained.

Role of extracellular calcium in in vitro uptake and intraphagocytic location of macrolides.

We compared the uptakes and intracellular locations of four 14-membered-ring macrolides (roxithromycin, dirithromycin, erythromycin, and erythromycylamine) in human polymorphonuclear neutrophils (PMNs) in vitro. Intracellular location was assessed by cell fractionation and uptake kinetics in cytoplasts (granule-poor PMNs). Trapping of dirithromycin within PMN granules (up to 80% at 30 min) was significantly more marked than the intracellular trapping of the other drugs (erythromycylamine, 45% +/- 5.1%; erythromycin, 42% +/- 3.7%; roxithromycin, 35% +/- 3.0%). A new finding was that, in the absence of extracellular calcium, the uptakes of all of the macrolides by PMNs and cytoplasts were significantly impaired, by about 50% (PMN) and 90% (cytoplasts). Furthermore, inorganic Ca2+ channel blockers inhibited macrolide uptake in a concentration-dependent manner, with 50% inhibitory concentrations of 1.6 to 2.0 mM and 29 to 35 microM, respectively, for Ni2+ and La3+. The intracellular distributions of the drugs were unchanged in the presence of Ni2+ and La3+ and in Ca(2+)-free medium supplemented with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The organic Ca2+ channel blocker nifedipine had no effect on macrolide uptake, whereas verapamil inhibited it in a time- and concentration-dependent manner. These data show the importance of extracellular Ca2+ in macrolide uptake by phagocytes and suggest a link with Ca2+ channels or a Ca2+ channel-operated mechanism.